Human mesenchymal stem/stromal cells (MSCs) have been widely used for immune system regulation and tissue repair. Human embryonic stem cells (hESCs) can be used as a reliable source for generating high-quality human MSCs. There are many methods to differentiate hESCs into MSCs. However, current methods are not able to conduct such differentiation in an efficient manner to produce a high yield of high purity MSCs.
Mesenchymal stem cells (MSCs) derived from adult mouse or human tissues such as bone marrow, umbilical cord and fat tissue are multipotent, i.e., capable of generating a variety of mature cell lineages including adipocytes, chondrocytes, osteoblast cells, neural lineage cells, myoblast, stromal cells and fibroblast, etc. These technologies have been well characterized and patented. For example, see Caplan et al., U.S. Pat. No. 5,486,359 (human mesenchymal stem cells).
However, the currently available adult tissue-derived MSCs have several pitfalls. First, the limited sources and varying quality of the donor tissues such as the bone marrow restrict the study and application of the MSCs and prevent the standardization of the MSCs as a medical product for large-scale clinical use. Second, the MSCs obtained from the adult tissues are highly mixed populations of cells, in which only a small portion of the cells have strong immunosuppressive effect. To obtain enough cell numbers for clinical use, in vitro expansion is necessary, which can decrease the immunosuppressive and homing abilities of MSCs (Javazon et al., 2004). Third, there are safety issues regarding to the use of adult-derived MSCs including malignant transformation (Wong, 2011) and potential transmission of infectious pathogens from donors.
To overcome these pitfalls, scientists have attempted to derive MSCs from hESCs via various methods. These methods involve either co-culture with the mouse OP9 cell line or handpicking plus the use of multiple cytokines and chemicals (Barbed et al., 2005; Chen et al., 2012; Liu et al., 2012; Sanchez et al., 2011). Recently, a TGFβ signaling inhibitor SB431542 has been used to differentiate hESCs into MSCs, which simplifies the procedures and improves the efficiency (Chen et al., 2012), but the yield and purity are quite low (see the below-described comparison tests.). In 2010, the inventors and Advanced Cell Technology developed another method to derive MSC from hemangioblast, which involved the use of many expensive cytokines and methylcellulose medium, but the derivation efficiency is also low using this method.
Currently known methods for differentiation of hESCs into MSCs are each characterized as having one or more serious shortcomings and weaknesses: Differentiation of MSCs from hESCs co-cultured with the OP9 stromal cells has the disadvantages of being time consuming, producing cells of low yield, low purity, and using animal feeder cells and undefined culture conditions (Barbed et al., 2005). Differentiation from outgrowing cells around replated embryoid bodies formed by hESCs has the disadvantages of being time consuming, producing cells in low yield, using undefined culture condition, and being an expensive method (Olivier et al., 2006). Differentiation from hESCs cultured on collagen-coated plates has the disadvantages of very low yield, undefined culture conditions, and being time consuming (Liu et al., 2012). Differentiation with hESCs treated with inhibitors of TGFβ signaling has the disadvantages including low purity of cells (per our tests), low cell yield, time consuming method, and low immunosuppressive effect of the cells that are produced (Chen et al., 2012; Sanchez et al., 2011). Thus, there is a need for an unlimited, safe, highly stable, efficient and consistent source of MSCs to use as a treatment and prophylactic for various diseases.
Multiple sclerosis (MS) is a chronic autoimmune disease caused by infiltration of peripheral immune cells into the central nervous system (CNS) through damaged blood-brain barrier (BBB) or blood-spinal cord barrier (BSCB), which causes inflammation of the myelin sheaths around neuronal axons, and causes demyelination and scarring of the axons (McFarland and Martin (2007)). According to the National Multiple Sclerosis Society of United States, there are more than 70 FDA-approved medications for the treatment of MS, including Avonex (IFNβ-1a), Betaseron (IFNβ-1b), Gilenya (a sphingosine 1-phosphate receptor modulator), Glatiramer acetate (or Copolymer 1), and Tysabri (humanized anti-α-integrin antibody). However, these offer only palliative relief and are associated with serious adverse effects including increased infection, heart attack, stroke, progressive multifocal leukoencephalopathy, arrhythmia, pain, depression, fatigue, macula edema, and erectile dysfunction (Johnston and So (2012): Weber et al. (2012)).
Transplantation of mesenchymal stromal/stem cells (MSCs) has emerged as a potentially attractive therapy due to their immunomodulatory and neuroregenerative effects (Auletta et al., (2012); Pittenger et al. (1999)) and potential ability to repair the blood-brain barrier (Chao et al. (2009); Mange et al. (2012)). MSCs are multipotent meaning they can generate a variety of call lineages including adipocyte, chondrocyte, osteoblast cells and neurons. They can be derived from fetal, neonatal, and adult tissues such as the amniotic membrane, umbilical cord, bone marrow, and adipose. MSCs have several unique advantages over current pharmacotherapies, as these cells can serve as carriers of multiple and potentially synergistic therapeutic factors, and can migrate to injured tissues to exert local effects through secretion of mediators and cell-cell contact (Uccelli and Prockop (2010a)). Importantly, MSCs have been found efficacious in the treatment of mice with experimental autoimmune encephalomyelitis (EAE), a well-recognized animal model of MS (Gordon et al., 2008a; Gordon et al. (2010); Morando et al. (2012); Peron et al. (2012); Zappia et al. (2005); Zhang et al. (2005)), as well as MS patients in clinical trials (Connick et al. (2012); Karussis et al. (2010); Mohyeddin Bonab et al. (2007); Yamout et al. (2010)). Xenogeneity does not appear problematic as both mouse and human bone marrow-derived MSC (BM-MSC) can attenuate disease progression of EAE mice (Gordon et al. (2008a); Gordon et al. (2010); Morando et al. (2012); Peron et al. (2012); Zappia et al. (2005); Zhang et al. (2005)). However, varying effects were reported on EAE mice treated with BM-MSC in different reports (Gordon et al. (2008a); Payne et al. (2012); Zappia et al. (2005); Zhang et al. (2005)). The efficacy of BM-MSC on treatment of the disease is questionable.
There is a strong need for an unlimited, safe, highly stable, efficient and consistent source of MSC to use as a treatment and prophylactic for these diseases as well as others. Disclosed herein are hES-T-MSCs derived from hESCs through a highly efficient differentiation method that meets these needs. Also disclosed herein are a microarray analysis and other analysis, where several key factors are identified that are differentially expressed in hES-T-MSC compared to BM-MSC and other hES-MSC differentiated through other methods.